Biomolecules having multiple attachment moieties for binding to a substrate surface

ABSTRACT

Methods of binding biomolecules to a substrate are provided that include contacting the biomolecule with a branched linking moiety to form a branched linking structure. The branched linking structure is then contacted with a binding moiety on the substrate to form a coupled substrate binding structure, thereby binding the biomolecule to the substrate. The biomolecule may contain a Lewis base or a nucleophile to react with a Lewis acid or electrophile in the branched linking moiety. Alternatively, the biomolecule may contain a Lewis acid or electrophile that can react with a Lewis base or nucleophile in the branched linking moiety. Additionally, the biomolecule can be bound to the substrate through a covalent or non-covalent bond.

FIELD OF THE INVENTION

This invention relates to attachment chemistries for bindingbiomolecules to a substrate surface. More particularly, this inventionrelates to attachment chemistries involving branched structures forproviding biomolecules having multiplicities of chemical bindingmoieties for binding the biomolecules to a substrate surface.

BACKGROUND OF THE INVENTION

The following description provides a summary of information relevant tothe present invention. It is not an admission that any of theinformation provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to the invention.

The immobilization of oligonucleotides on substrates is an important andnecessary step for many applications such as DNA chip technology,surface plasmon resonance experiments, or other biosensor applications.Classically, oligonucleotides are immobilized onto substrates bymodification of the 3′- or 5′-end with one reactive group e.g. an amine,thiol or aldehyde (covalent attachment) or group forming stablecomplexes e.g. biotin, phenylboronic acid etc. (noncovalent attachment).The modified oligonucleotides are then addressed to the location wherethe immobilization is desired and reacted with an appropriate functionalgroup such as an aldehyde, maleimide, hydrazide etc. or complexed with abinding molecule such as streptavidin, etc. The addressing to specificlocations on a substrate can be done by spotting (pin or dropdeposition), by electronic addressing, or by a variety of otherprocesses. In some cases the reaction for the immobilization is slow andrequires long (overnight) incubation of the oligonucleotides on thesubstrate. These immobilization reactions may also be reversible,resulting in the release of the biomolecule over time.

In other instances, dendrimeric structures on biomolecules has beendescribed (e.g., WO 99/10362, WO 96/19240, and WO 99/43287), but the useof the dendrimeric structures have been directed toward providing signalsites such as for detection while the biomolecule itself is simplyattached to a substrate using classical means.

In contrast thereto, the present invention describes an improved processfor immobilization of biomolecules using oligonucleotides containingmultiple reactive sites, i.e. nucleophiles, electrophiles, and Lewisacids or bases. The advantage of this approach is a higher rate ofimmobilization, a higher stability of the attachment, and the potentialto obtain higher amounts of immobilized oligonucleotide onto thesubstrate surface. These gains are independent of the approach used forthe immobilization. Oligonucleotides with multiple attachment sites canbe obtained with both covalent and noncovalent attachment chemistries.

Furthermore the present invention describes the preparation ofoligonucleotides containing one or more hydrazides. Hydrazides arenucleophilic reactive groups that can be used for any type ofconjugation reaction. They can react, for example, with electrophilicaldehydes forming hydrazones (which can be further stabilized byreduction) and with active esters forming stable covalent linkages, seeFIG. 18. This chemistry can be used for attaching fluorophores, proteinsor peptides, reporter groups and other oligomers to oligonucleotides.The reactions of hydrazides can also be used for the immobilization ofbiomolecules onto substrates. Such hydrazide modified oligonucleotideshave not been previously described.

The advantages of this invention within the scope of this descriptionare numerous. For example, this invention uses a short reaction time,allows for multiple binding sites per bound entity, provides forstability to a relatively broad pH range, and provides for thecapability of attachment under both anhydrous or aqueous conditionsthereby providing an improved method for attaching molecules to anysolid phase surface for any applicable use. The invention is useful forsolid phase synthesis and/or synthesis of small molecule libraries suchas biomolecules including, but not limited to, DNA, RNA, PNA, p-RNA(pyranosyl-RNA), and peptides. The invention is also useful foranalytical techniques that require an immobilized reagent such as,without limitation, hybridization based assays, diagnostics, genesequence identification and the like.

SUMMARY OF THE INVENTION

In a first embodiment of the invention, biomolecules are provided havinga multiplicity of branched or dendrimeric moieties for connectingthereto functional or reactive moieties for binding to a substratesurface.

The use of oligonucleotides with multiple reactive sites or complexingagents within one oligonucleotide offers significant advantages to thisimmobilization process. First, it increases the speed of theimmobilization process. One reason for this effect is that chance for aninitial contact between the attachment partners by diffusion is higherwhen one oligonucleotide bears multiple reactive sites. Additionally,the oligonucleotide can be immobilized via secondary and multiplecovalent or noncovalent linkages which are formed after (or simultaneouswith) the primary linkage. The formation of these secondary linkages isthen an intramolecular process that is kinetically favored to theintermolecular primary linkage formation. This is another reason for thehigher immobilization rate.

Second, the overall stability of the attachment increases as multiplelinkages are formed between the oligonucleotide and the substrate whichis independent of the approach used to bring the biomolecule intocontact with the substrate.

The formation of multiple noncovalent complexes results in a higheroverall stability of the complex between the oligonucleotide and thesubstrate allowing the use of low affinity complex builders for astabile immobilization. Some of the frequently applied immobilizationchemistries for oligonucleotides are reversible (e.g. the Schiffs baseformation between amines and aldehydes) and require a subsequentstabilization step e.g. by reduction with NaCNBH₃. For these reversiblereactions the immobilization via multiple linkages is beneficial sinceit leads in sum to a higher stability of the intermediates formed priorto the stabilization reaction. In some cases the gain in stability isgreat enough that the stabilization reaction becomes unnecessary.

Third, the use of oligonucleotides with multiple attachment sites allowsthe production of substrates with higher oligonucleotide loading.Usually the reactive sites on the substrate are in large excess to theoligonucleotides and the improved attachment due to multiple attachmentmoieties can lead to better use of the available sites on the substrate.

In another embodiment, the multiplicity of reactive binding moietiesprovided on the biomolecules may allow the biomolecules to bind, eitherin a covalent or a noncovalent manner, to the substrate surface. Withrespect to noncovalent binding, the multiplicity of binding moieties maycomprise chemical moieties such as biotin, streptavidin, phenyl boronicacid (PBA), and salicyl hydroxamic acid (SHA). With respect to covalentbinding, the multiplicity of binding moieties may comprise the use ofreactive hydrazide structures. Such structures may be either branched orunbranched thereby allowing for great versatility in the level ofpossible binding moieties available. Thus, not only are the biomoleculesprovided with dendritic branching structures, but the reactive bindingmoieties themselves may also be branched such that each branch has areactive hydrazide element for use in binding the biomolecule to asubstrate surface.

In another embodiment, the multiplicity of binding moieties on thebiomolecule provides a means whereby biomolecules attached to asubstrate surface comprising an electronically addressable microchip areprotected from inadvertent removal from the attachment site on themicrochip caused by high voltage and current resulting from electronicbiasing of the microchip electrode. Thus, in a preferred embodiment, themultiple attachment scheme of the current invention provides for bindingof biomolecules to the substrate capable of withstanding currentdensities of at least 4 mA/cm².

In still another embodiment, the invention provides for a method ofadding reactive binding moieties to the dendritic structures attached tothe biomolecules such that the addition may occur in a single reactionstep.

In still another embodiment, the invention provides a composition ofmatter comprising new chemical modifications of oligonucleotidescontaining one or multiple hydrazides and thereby the building blocks(e.g. phosphoramidites) for the generation of modified oligonucleotidesthereof. These hydrazides comprise reactive groups and can be used forthe conjugation of oligonucleotides to fluorophores or other smallmolecules, to peptides, proteins or antibodies, or to substratesurfaces.

In still another embodiment the attachment scheme can be applied tosurface synthesis of biomolecules and analytical applications requiringsurface immobilization of compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the classic approach for immobilizingoligonucleotides on planar substrates. Generally, a single reactivegroup is used to bind the oligomer to the substrate surface.

FIG. 2 is a schematic depiction of the immobilization approach of theinvention wherein the biomolecule has a multiplicity of attachment orbinding moieties that can participate in the covalent or noncovalentbinding of the biomolecule to the substrate.

FIG. 3 is a more detailed view of one example of a nucleic acid strandattached to a multiplicity of reactive moieties for binding to asubstrate surface. In this example, the chemical structures of branchedphosphoramidites are added in multiple fashion to create a dendrimericstructure attached to the biomolecule.

FIG. 4 shows a series of chemical steps to produce a structure having adendrimeric structure containing phenyl boronic acid (PBA) attachmentmoieties.

FIGS. 5A and B show a series of chemical steps to produce chemicalstructures comprising oligonucleotide biomolecules having either four(A) or eight (B) binding moieties for noncovalent binding of thebiomolecule to a substrate surface.

FIGS. 6A-C illustrate synthetic steps using phosphoramidites to producebiomolecules having multiple reactive sites. These moieties containester groups that are converted into hydrazides during the deprotectionof the oligonucleotides with hydrazine.

FIG. 7 shows chemical structures A and B comprising biomolecules havingdirect attachment of hydrazide moieties for use in immobilizing thebiomolecule through a covalent bond to the substrate.

FIGS. 8A-D show chemical syntheses to produce structures having multiplebinding moieties. In (A) a branched phosphoramidite is added to anoligonucleotide which is further modified with a bifunctionalphosphoramidite followed by deprotection with of diethylamine/CH2Cl2 andhydrazine to generate a binding moiety having four hydrazide groups forbinding a substrate. In (B) a reaction scheme similar to (A) is providedresulting in six hydrazide binding moieties. In (C) the sequential useof two different branched phosphoramidites results in 16 hydrazidebinding moieties per biomolecule. In (D) a branched phosphoramidite isused in two steps to form a dendrimeric structure followed by aphosphoramidite and hydrazine treatment to result in 4 hydrazide bindingmoieties per biomolecule.

FIGS. 9A-C show three schemes of which A and B show steps for making anovel phosphoramidite for incorporation into an oligonucleotidebiomolecule. FIG. 9C shows a scheme wherein the hydrazide-labeledoligonucleotide can react with an activated-ester monomer and used toform a substrate for immobilization of biomolecules.

FIGS. 10A-C are graphs of three separate HPLC traces of the reactionmixture for coupling hydrazide-labeled oligo to an activated estermonomer such as that shown in Scheme 3 of FIG. 9.

FIG. 11 is a graph that shows reaction rates for attachment ofmulti-labeled biomolecules. The hydrazide/N-hydroxysuccinimidyl (NHS)ester binding occurred at a rate measurably above that of other covalentbinding systems and near to that of two noncovalent systems.

FIG. 12 is a graph that shows that covalent attachment of the labeledhydrazide oligo is dependent on the amount of activated ester in thesubstrate surface.

FIGS. 13 and 14 are graphs showing the proficiency of covalentattachment for either NHS or N-hydroxy-sulfosuccinimidyl (Sulfo-NHS)ester modified substrate surfaces respectively. The graphs show thespecific and nonspecific fluorescent intensity from labeled oligomersattached to the electrodes over a range of applied currents.

FIG. 15 is a graph that shows that multiple binding of hydrazidemoieties provides higher level of detection of the biomolecule on thesubstrate.

FIG. 16 is a graph that shows results of an electronic reverse dot blotin which hybridization was completed only on those sites containing ahydrazide-modified oligonucleotide (ATA5). The capture probes werespecifically bound to an activated-ester-containing substrate underappropriate electronic conditions. The nonspecific captures without ahydrazide do not react with the activated ester and are thereforeunavailable for hybridization.

FIG. 17 shows the synthesis of a hydrazide modified oligonucleotidefollowing two distinct protocols A and B. In A, a protected hydrazidephosphoramidite is used to modify the oligomer, which is thendeprotected. In B, an ester phosphoramidite is used to modify theoligomer, which is then reacted with hydrazine.

FIG. 18 shows a schematic illustrating the various functional groupswhich are capable of reacting with hydrazide modified oligomers.

FIGS. 19 and 20 are examples of hydrazide oligomers condensing withaldehydes.

FIG. 21 shows the recorded mean fluorescent intensity (MFI) values foroligomers labeled with 3, 4, and 8 phenylboronic acids per oligo. Theattached oligomers were subjected to vigorous washing conditions tomonitor the stability of the attachment system.

FIG. 22 shows a schematic illustrating the dynamic equilibrium andstability of dendrimeric hydrazides onto an aldehyde rich permeationlayer. The oligomer, displayed with four hydrazide moieties, iselectronically loaded onto an aldehyde rich permeation layer resultingin multiple hydrazone linkages. In this particular example, the linkagesindividually are susceptible to hydrolysis. The stability gained withthe use of multiple attachment sites allows for hydrolysis of somehydrazones while others remain intact. The hydrazide, tethered throughneighboring hydrazone attachment sites, is incapable of diffusion and istherefore retained within the aldehyde rich permeation layer capable ofre-establishing the linkage.

FIG. 23 is a graph showing the attachment of dendrimeric oligomers of 1,2, 4, and 8 hydrazides onto glyoxyl agarose permeation layers coupledvia hydrazone linkage(s).

FIG. 24 is a graph showing the attachment of dendrimeric oligomers ontoan acetal modified hydrogel. The acetal moieties require hydrolysis withacid to generate aldehydes for covalent attachment capabilities.

FIG. 25 illustrates the use and improved binding of hydrazide oligomerson Surmodics 3D Link™ Amine Binding Slides at various concentrations.FIG. 25B are the actual fluorescent photoimages of the oligomers boundto the glass slide, their binding levels are displayed graphically inFIG. 25A.

FIG. 26 displays the nonspecific attachment binding levels to theSurmodics slide used in FIG. 25. FIG. 26B are the actual fluorescentphotoimages of the oligomers bound to the glass slide, their bindinglevels are displayed graphically in FIG. 26A.

FIG. 27A shows the applicable pH range in which the hydrazide oligomersare capable of successful immobilization to a solid support; FIG. 27Bdisplays the improved sensitivity of a hydrazide oligomer over astandard amine modified oligomer being detectable at lowerconcentrations.

FIG. 28 illustrates one example of how a branched or unbranchedhydrazide modified oligomer can be easily modified to an alternativeattachment system. In this particular example a branched oligomer withsix hydrazides is modified with p-formylphenylboronic acid to afford abranched PBA attachment probe.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the specific embodiments of the invention, biomoleculesare provided having a multiplicity of substrate surface bindingmoieties.

By “biomolecule” is meant a biologically relevant molecule that is usedto contact molecular entities in a test sample. Generally, theseinclude, at least in part, molecules such as nucleic acids, including asingle nucleic acid, oligonucleotides and polynucleotides, DNAs, RNAs,CNAs (cyclohexyl nucleic acids), p-MeNAs (methyl or methoxy phosphatenucleic acids), proteins, peptides, enzymes, and antibodies attached tochemical moieties for binding the biomolecule to a substrate surface.Biomolecules also include unnatural or synthetic molecules structurallyderived from the naturally occurring molecules such as peptide nucleicacids (PNAs) or p-RNAs (pyranosyl RNAs) attached to chemical moietiesfor binding the biomolecule to a substrate surface. Having such abinding moiety, biomolecules may also be referred to as “derivatizedbiomolecules”. Such biomolecules therefore also include oligonucleotidescontaining oxidized ribose, amine terminations, or any entity of thewell known bioconjugate pairs as outlined by Hermanson (Hermanson, G. T.Bioconjugate Techniques copyright 1996, Academic Press, San Diego,Calif.) herein incorporated by reference, and/or alternative nucleicacid structures such as pRNAs (in reference to pRNAs as described inco-pending application Ser. No. 09/374,338 filed Aug. 13, 1999 hereinincorporated by reference). Generally, attachment of the chemicalmoieties to the biomolecules comprises a covalent bond. With respect toattachment of derivatized biomolecules to a substrate surface, suchattachment may use either a covalent or a noncovalent bond.

By “polymer” is generally meant macromolecules assembled from thesuccessive linkage of a large number of smaller molecules generallyreferred to as monomers as recognized by one skilled in the art (for amore detailed description see Odian, G. Principles of Polymerization,Third Edition copyright 1991 John Wiley and Sons Inc., New York, N.Y.).In a preferred embodiment, a homogeneous polymer may be composed of asingle type of monomer, while a heterogeneous polymer is be composed ofmore than one type of monomer. In another preferred embodiment,formation of a polymer can be initiated by thermal decomposition ofinitiators (e.g. AIBN, benzoyl peroxide), photolytic cleavage ofinitiators (e.g. UV initiation of Daracur 4265), redox reactions (e.g.cerium (IV) sulfate), ionizing radiation (e.g. α, β, γ or X-rays),plasma initiation (e.g. Argon, Nitrogen, Oxygen), or electrolyticinitiation using tetrabutylammonium perchlorate in which thepolymerization occurs only over a preselected site using an electriccurrent (Samal, S. K.; Nayak, B. J. Polym. Sci. Polym. Chem. Ed. 1988,21, 1035.)

By “binding moiety” is generally meant any chemical moiety utilized inthe generation of attachment of biomolecules to a substrate surface. Abinding moiety may be contained on a biomolecule or contained on asubstrate surface. Table 1 Binding Moieties provides a list of bindingmoieties used.

TABLE 1 Binding Moieties Functional Group or Chemical Structure Name

Alcohol

Ether

Primary amine

Substituted amine

Hydrazine

Sulfhydryl

Epoxide

Aziridine

Vinyl

Allyl

Aldehyde

Ketone

Acetal

disulfide

Ester

Carboxylic Acid

Amide

Monosubstituted Amide

Disubstituted Amide

Bromo- or Iodo-acetamide

Hydrazide

Thioester

(sulfonated)-N-Hydroxy succinimidyl ester

Azlactone, an activated ester X—N═C═O Isocyanate X—N═C═S Isothiocyanate

Acyl azide

Carbonates

phosphoramidites Symbols: X = a biomolecule or substrate/solid support;R, R₁, R₂, R₃ = organic carbon moieties unless otherwise indicated.

By “Lewis Base” is generally meant any chemical moiety capable ofdonating a pair of electrons to an electron deficient center. In apreferred embodiment, a Lewis Base is more specifically referred to as a“nucleophile” in which a reactive center donates a pair of electrons tocarbon resulting in a covalent bond between the reactive center and thecarbon as recognized by one skilled in the art (For an expandeddefinition see: Smith, M. B. Organic Synthesis copyright 1994 McGrawHill Inc., New York, N.Y., or any organic chemistry textbook).

By “Lewis acid” is generally meant any electron deficient chemicalmoiety capable of receiving a pair of electrons. By “electrophile” isgenerally meant the specific case in which the Lewis Acid is carbon, asrecognized by one skilled in the art (For an expanded definition see:Smith, M. B. Organic Synthesis copyright 1994 McGraw Hill Inc., NewYork, N.Y., or any organic chemistry textbook). In a preferredembodiment, as an example, salicylic hydroxamic acid is capable ofacting as a Lewis base donating a pair of electrons to boron, a Lewisacid, of phenyl boronic acid resulting in a noncovalent linkage. In yetanother preferred embodiment, as an example, hydrazide is capable ofacting as a nucleophile donating a pair of electrons to the reactivecarbon center of an NHS ester, an electrophile, forming a covalentlinkage to said carbon center.

By “branched linking moiety” is generally meant any chemical specieswhich is capable of coupling through a specific reactive moiety to abiomolecule and is also capable of further attachment to more than onemolecule through alternative reactive centers. In a preferredembodiment, a branched linking moiety is a phosphoramidite of whichexamples are shown in Table 2, Entries 1-4. In these examples, thephosphorus acts as the reactive moiety while the esters of entries 1, 2,and 3 and the protected alcohols of 4 are alternative reactive centers.

By “branched linking structure” is generally meant a biomoleculeresulting from treatment of a biomolecule with a branched linkingmoiety. The alternative reactive centers of the branched linking moietyare now contained within the branched linking structure. In a preferredembodiment, as an example, a branched linking structure is representedby entry 5 of Table 2 in which the biomolecule shown is the result oftreating a biomolecule with a branched linking moiety, specifically thecompound displayed in entry 4 of Table 2. In another preferredembodiment the branching linking structure is capable of being combinedin a homogeneous series in which a biomolecule is modified with abranching linking moiety, which in turn is further modified by the samebranched linking moiety through the alternative reactive centers of theresultant branched linking structure, generating a new branched linkingstructure. This construction of larger branched linking structures bymeans of a series of linkages of a branched linking moiety can befurther continued as shown in Table 2, Entries 6-8 In yet anotherembodiment, the branching linking moieties are capable of being combinedin a heterogeneous series in which a biomolecule is modified with abranching linking moiety, which in turn is further modified by adifferent branched linking moiety through the alternative reactivecenters of the initial branched linking moiety, generating a newbranched linking structure. This construction of larger branched linkingstructures by means of a series of linkages of branched linking moietiescan be further continued as shown in Table 2, Entries 9-12.

TABLE 2 Branched Linking Moieties and Branched Linking Structures Nameor class of Entry Chemical Structure compound 1

Branched Linking Moiety: Diethyl 5-{[(2-cyanoethoxy) (diisopropylamino)phosphanyloxy] methyl}isophthalate; Compound 1c; a diesterphosphoramidite 2

Branched Linking Moiety: Diethyl 3-[(2-cyanoethoxy) (diisopropylamino)phosphanyloxy] glutarate; a branched diester phosphoramidite. 3

Branched Linking Moiety: Dimethyl 3,3′-(2-{[(2- cyanoethoxy)(diisopropylamino) phosphanyloxy] methyl}-2-{[2- (methoxycarbonyl)ethoxy]methyl} propane-1,3- diylbisoxy) dipropionate; Compound ld; atri-ester phosphoramidite 4

Branched Linking Moiety: 1,3-bis-((di p-methoxyphenyl)- phenylmethoxy)-2-propyl O-2- cyanoethyl-N,N- diisopropylamino phosphoramidite; asymmetrically branched phosphoramidite. DMT = di-(p- methoxyphenyl)-phenylmethyl 5

First generation branched linking structure in which the alternativereactive species is an alcohol (R = H) or a protected alcohol (R = DMT).6

Second generation homogeneous branched linking structure in which thealternative reacting species is an alcohol (R = H) or a protectedalcohol (R = DMT). 7

Third generation homogeneous branched linking structure in which thealternative reacting species is an alcohol (R = H) or a protectedalcohol (R = DMT). 8

Fourth generation homogeneous branched linking structure in which thealternative reacting species is an alcohol (R = H) or a protectedalcohol (R = DMT). 9

Second generation heterogeneous branched linking structure. 10

Second generation heterogeneous branched linking structure. 11

Second generation heterogeneous branched linking structure. 12

Third generation heterogeneous branched linking structure.

By “substrate” is generally meant any material whose surface containsmoieties with which the multiple reactive binding moieties of thebiomolecules may couple. This substrate can be, among others, a glassslide, a functionalized glass slide, a chemically activated microchipsurface, a surface covered with a single or multiple layers of reactivemolecules, or a surface covered with a polymer having moieties withwhich the multiple reactive binding moieties of the biomolecules mayreact. In a preferred embodiment, a substrate surface is a permeationlayer of an electronically addressable microchip. In a preferredembodiment, the functional, chemically active, or reactive moieties of asubstrate are selected from (but not limited to) the functional groupslisted in Table 1.

By “precursor” is generally meant any reactive moiety which can betransformed to an alternative reactive moiety with treatment of one ormore chemical reagents. In a preferred embodiment, as an example, thethree ester moieties of 1d, (Entry 3 of Table 2) are precursors tohydrazides. They are transformed to a hydrazide moiety with thetreatment of hydrazine.

By “protected” is generally meant blocking the reactivity of a reactivemoiety with one or more reagents while a chemical reaction can becarried out at an alternative reactive site of the same compound withoutobstruction or complication from the initial reactive moiety. Uponcompletion of the transformation at the alternative reactive site theprotecting group of the reactive moiety can be removed, unblocking thereactive center. In a preferred embodiment, a protected moiety is aspecific type of precursor. In yet another preferred embodiment, as anexample, the hydrazide moiety of 1a of FIG. 9A is protected with atrityl group. Upon addition of 1a to a biomolecule the trityl group ischemically removed deprotecting the hydrazide functionality.

By “activatable” is generally meant any functional group which iscapable of undergoing a transformation to a reactive moiety when treatedwith one or more chemical reagents. By “activated” is meant a functionalgroup which has undergone such a transformation to a reactive moiety. Ina preferred embodiment, an activatable moiety can be a protected moietyor a precursor. In yet another preferred embodiment, the functionalgroup is generally considered benign, unreactive, or incapable ofbinding to a substrate or biomolecule. Upon treatment with one or morechemical reagents, the functional group is transformed to a moietycapable of binding to a substrate or biomolecule. In a preferredembodiment, as an example, the ester groups of the compounds listed inTable 2 Entries 1-3 are transformed to hydrazides with treatment withhydrazine. In yet another preferred embodiment, as an example, asubstrate containing acetal groups is generally considered to beunreactive. Upon treatment with an acidic source, the acetals aretransformed to aldehydes which are capable of binding to hydrazidemodified biomolecules.

By “microarray” is generally meant a geometric arrangement of aplurality of locations containing defined biomolecules, such individuallocations being restricted to linear dimensions of 1 mm or less.Microarrays include an electronically addressable microarray such as anarray designated the “APEX chip” as in U.S. Pat. No. 5,632,957 hereinincorporated by reference.

FIG. 2 shows a basic schematic for the scheme of the invention whereinbiomolecules are bound to a substrate surface through a multiplicity ofattachment moieties. The multiples of attachment moieties may beprovided to a biomolecule using the following methods. Each of theseapproaches is compatible with standard solid phase synthesis ofbiomolecules comprising oligonucleotides.

1. Preparation of Oligonucleotides with Multiple Attachment Sites1.1 Oligonucleotide Synthesis with Branching Phosphoramidites:

Branched biomolecule (e.g. oligonucleotides) structures having branchedphosphoramidites are commercially available (Chemgenes, Ashland, Mass.;Glenn Research, Sterling, Va.). After one or more consecutive couplingsof such branching amidites in the solid-phase oligonucleotide synthesis(FIGS. 5A and B), oligonucleotides with two or more terminal hydroxylgroups are generated. Any other building block introducing branches intothe oligonucleotide can be applied here in a similar manner. Thesehydroxyl groups can be reacted with a second type of phosphoramidite togenerate the reactive group (i.e. the binding moiety) for the attachmentof the biomolecule to the substrate. This phosphoramidite can be chosenfrom several available amidites such as biotin amidites (e.g. GlennResearch, Cat No. 10595002), amino modifiers (e.g. Glenn Research, Cat.No. 10190602), thiol modifiers (e.g. Glenn Research, Cat. No. 10192602),phenylboronic acid amidites (Prolinx, Bothell, Wash.) and others.Further, phosphoramidites containing hydrazides in a protected orprecursor form (FIG. 9A) can be used. The result is an oligonucleotidehaving two or more (preferably 2 to 8) reactive groups.

1.2 Direct Introduction of Synthons Having More than One Reactive Groupfor Attachment:

Alternatively, biomolecules with multiple attachment sites can beobtained by the coupling of special phosphoramidites. These amidites cancontain in a protected or precursor from more than one reactive groupfor the immobilization at the substrate. The reactive group in branchedamidites can be again one of the known functionalities such as aminogroups, thiols, aldehydes, or hydrazides. Examples for such amidites areshown in FIGS. 6A-C.

1.3 Combined Approach:

A third approach for the synthesis of biomolecules with multiplereactive groups is the combination of the coupling of branching amiditesand amidites with multiple reactive sites (FIGS. 8A-C).

In a particularly preferred embodiment, biomolecules are provided havinga tethered hydrazide for attachment to a substrate surface through acovalent bond. In this embodiment, NHS and Sulfo-NHS and other moietiesmay be used as a means of activating a substrate or any other type ofbiomolecule and coupling to biomolecules or even solid surfaces. In theapplication of the present invention, such attachment provides a novelmeans whereby biomolecule attachment may be carried out and provide forresistance against damage to tethered biomolecules caused by the extremereaction conditions associated with electronic addressing of anelectronic microchip. Thus, the hydrazide chemistry and multipleattachment scheme of the present invention fulfills requirements forsurvivability in the environment of an electronic system whichrequirements include a need for water solubility of the biomolecule,stability to water of the biomolecule and its coupling pair on theimmobilizing substrate, and functionality to a pH of approximately pH 4.

The methods by which hydrazide binding moieties were added and utilizedin the present invention are provided in the following examples. Theseexamples show site specific covalent attachment of a biomoleculecomprising an oligonucleotide in which attachment is accomplished withelectronic concentration of a hydrazide-modified oligo onto anN-hydroxysuccinimidyl (NHS) modified polyacrylamide permeation layerabove an electronically addressable microarray. The hydrazide moiety ofthe oligomer displaces the NHS ester forming a bishydrazide linkage.These examples therefore show 1.) Synthesis of the novel hydrazidephosphoramidite (e.g., compound 1) as shown in Example 1 (FIG. 9) andsuccessful incorporation of these amidites onto synthetic oligomersusing standard synthetic procedures; 2.) Preparation of N-Hydroxy- orN-hydroxysulfo-succinimidyl modified permeation layer; and 3.) Atwo-layer permeation layer above the electronically addressablemicroarray in which the activated monomers are incorporated into onlythe top layer.

Unless otherwise indicated, all reactions were magnetically stirred.Reagents were obtained in analytical grade from Aldrich Chemical Company(Milwaukee Wis.) and solvents from Riedel. Column Chromatography isaccomplished using silica gel 60 (Merck, 230-400 mesh). Melting pointsare uncorrected. IR Spectra are measured on a Perkin Elmer Paragon 1000FT-IR equipped with a Graseby Specac 10500 ATR unit. ¹H-NMR spectra arerecorded at 400 MHz; ¹³C spectra at 100 MHz and ³¹P at 162 MHz with aBruker DRX 400 spectrometer. ¹H chemical shifts are reported in units ofδ using TMS as internal standard, and coupling constants are reported inunits of Hz. ESI Mass spectra are recorded on a Finnigan LCQ instrumentin negative ionization mode.

Example 1 Experiment 1.1 Synthesis ofN-Triphenylmethyl-6-hydroxycapronic acid hydrazide, (compound 5, FIG.9A)

To a solution of 6.2 g (20 mmol) of tritylhydrazine hydrochloride (3a)in 200 ml of THF was added 2.22 g (22 mmol, 1.1 eq) triethylamine. Thesolution was stirred at room temperature (rt) for 15 min, filtered,concentrated to afford compound 3, then treated with 2.29 g (20 mmol, 1eq) of ε-caprolactone (compound 4). The mixture is heated to 65° C. for5 h the cooled to rt for 18 h. The precipitate was collected andrecrystallized from ethyl acetate to afford 3.55 g (45%) of a whitepowder (compound 5): ¹H-NMR 7.49-7.47 (m, 5H), 7.35-7.10 (m, 10H), 6.55(d, J=7.52, 1H), 5.55 (d, J=7.25, 1H), 3.54 (t, J=6.45, 2H), 1.87 (t,J=7.25, 2H), 1.62 (bs, 1H), 1.57-1.34 (m, 4H), 1.27-1.11 (m, 2H).

Experiment 1.2 Synthesis of6-[(2Cyanoethoxy)(diisopropylamino)phosphanyloxy]-N′-tritylhexanohydrazide(compound 1a, FIG. 9A)

To a solution of 3.0 g (7.7 mmol) N-triphenylmethyl-6-hydroxycapronichydrazide (compound 5) in 50 ml dry dichloromethane at rt was slowlyadded 4.0 g (31 mmol, 4 eq) of N-ethyldiisopropyl amine and 2.01 g (8.5mmol, 1.1 eq) of chloro(diisopropylamino)-β-cyanoethoxyphosphine(compound 6) over 15 min. Upon complete addition, the reaction wasstirred for 1 h, concentrated, and chromatographed (ethylacetate/n-heptane ⅔ with 0.2% triethylamine) to afford 3.19 g (70%) of1a as a pale yellow foam.

¹H-NMR: 7.49-7.46 (m, 5H), 7.34-7.20 (m, 10H), 6.57 (d, J=7.2, 1H), 5.57(d, J=7.5, 1H), 3.85-3.74 (m, 2H), 3.62-3.48 (m, 4H), 2.62-2.59 (m, 2H), 1.88-1.84 (m, 2H), 1.53-1.33 (m, 4H), 1.27-1.13 (m, 14H); ³¹P-NMR(CDCl₃): δ=147.97.

Experiment 1.3 Preparation of Ethyl6-[(2-cyanoethoxy)(diisoprooylamino)phosphanyloxy]hexanoate (compound1b, FIG. 9B. Scheme 2)

To a solution of 1.65 g (10 mmol) of ethyl 6-hydroxyhexanoate (compound7) in 30 ml dichloromethane at rt are slowly added 5.17 g (40 mmol, 4eq) of N-ethyldiisopropyl amine and 2.6 g (11 mmol, 1.1 eq) of compound6 over 15 min. Upon complete addition, the reaction was further stirredfor 15 min, concentrated, and chromatographed (ethyl acetate/n-heptane ¼with 0.2% triethylamine) to afford 2.47 g (69%) of compound 1b as clearoil: ¹H-NMR 4.12 (q, J=7.25, 2H), 3.90-3.77 (m, 2H), 3.75-3.55 (m, 4H),2.64 (t, J=6.44, 2H), 2.30 (t, J=7.25, 2H), 1.69-1.59 (m, 4H), 1.44-1.34(m, 2H), 1.25 (t, J=7.25, 3H), 1.20-1.12 (m, 12H); ³¹P-NMR (CDCl₃):δ=148.01.

Experiment 1.4 Preparation of ester phosphoramidite: Diethyl5-{[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]methyl}isophthalate(Compound 1c FIG. 6B)

To a solution of 1.29 g (5 mmol) diethyl 5-(hydroxymethyl)isophthalate[252.27] (98%, Aldrich; CAS 181425-91-2) in 20 ml dry dichloromethane atRT are added 2.59 g (40 mmol, 4 eq) N-ethyldiisopropyl amine [129.25]and 1.3 g (11 mmol, 1.1 eq) 2-cyanoethylN,N-diisopropyl-chloro-phosphoramidite [236.68] (Aldrich; CAS89992-70-1) over 15 min with stirring. The mixture was concentrated andsalts were precipitated with 30 mL ethyl acetate/n-heptane (2:3). Thehydrochloride precipitate is filtered; the filtrate is concentrated anddirectly applied to a chromatography column. Elution with ethylacetate/n-heptane (1:4) containing few drops triethylamine afforded 1.6g (70%) 1c as a colorless oil. C₂₂H₃₃N₂O₆P; ¹H-NMR 8.59 (m, 1H, arom.),8.21 (m, 2H, arom.), 4.87-4.75 (m, 2H, CH₂ cyanoethyl), 4.41 (q, J=6.98Hz, 4H, CH₂ ethyl), 3.95-3.80 (m, 2H, 2×CH I—Pr), 3.74-3.61 (m, 2H, CH₂cyanoethyl), 2.66 (t, J (P,H)=6.45 Hz, 2H, O—CH₂-arom), 1.41 (t, J=6H,2×CH₃ ethyl), 1.23-1.20 (m, 12H, CH₃, I—Pr); ³¹P-NMR (CDCl₃): δ=149.94;¹³C-NMR (CDCl₃): δ=165.8 (C═O), 140.2 (C—CH₂—O—P), 132.1 (2×C arom.),131.1 (2×C—H arom), 129.7 (C H arom), 117.6 (CN), 64.7 (P—O—CH₂-arom),61.4 (2×CH₂ ethyl), 58.6 (O-CH₂—CH₂—CN), 43.4 (2×C—H I—Pr), 24.7 (4×CH₃I—Pr), 20.5 (O—CH₂—CH₂—CN), 14.4 (CH₃ ethyl); HRMS 453.2156([M+H]⁺C₂₂H₃₄N₂O₆P requires 453.21545).

Experiment 1.5 Synthesis of Dimethyl3,3′-(2-{[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]methyl}-2-{[2-(methoxycarbonyl)ethoxy]methyl}propane-1,3-diylbisoxy)dipropionate(compound 1d FIG. 8B)

To a solution of 300 mg (0.760 mmol)Tris-2,2,2-{[(methoxycarbonyl)ethoxy]methyl}ethanol (CAS 169744-28-9;(Coutts, S.; Jones, D. S.; Livingston, D. A.; Yu, L.: 1995,Chemically-defined non-polymeric valency platform molecules andconjugates thereof, European patent application EP 0642798A2) in 2 mldry dichloromethane at RT are added two drops of a 0.4 M solution of1H-Tetrazole in dry acetonitrile (standard activator solution from solidphase DNA synthesis) and 274 mg (0.91 mmol; 1.1 eq) 2-cyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (Aldrich; CAS 102691-36-1)and stirred at RT until TLC shows complete consumption of the startingmaterial (3 h). The solvent is removed in vacuo and the residue ispurified by silica gel chromatography. Elution with ethylacetate/n-heptane (2:3) containing few drops triethylamine afforded 240mg (53%) of 1d as colorless oil. C₂₆H₄₇N₂O₁₁P ¹H-NMR (CDCl₃): 3.88-3.71(m, 2H, C—H), 3.68 (s, 9H, CH₃ ester), 3.65 (t, J=6.45 6H, 3×CH₂—O),3.62-3.47 (m, 4H, 2×CH₂), 3.36 (s, 6H, 3×C—CH₂—O), 2.63 (t, J=7.25 Hz,2H, C—CH₂—O—P), 2.54 (t, J=6.45 Hz, 6H, —CH₂—COOR), 1.19-1.16 (m, 12H,CH₃ iPr); ³¹P-NMR (CDCl₃): δ=148.6; HRMS: 595.2999 ([M+H]⁺C₂₆H₄₈N₂O₁₁Prequires 595.29957)

Experiment 1.6 Synthesis of Oligonucleotides with trityl ProtectedHydrazide Amidites (e.g. Compound 1: See Also FIG. 17A)

Oligonucleotides are synthesized using solid phase phosphoramiditechemistry on an automated oligonucleotide synthesizer. Thephosphoramidite with the protected hydrazide is applied as 0.1 Msolution in acetonitrile and coupled at the desired location in thesequence using standard activated reagents and coupling times.

The CPG bound oligo (1 mmol) is placed in a 1.5 ml test tube and treatedwith 2.0 ml conc. NH₄OH. After 2 h at 55° C. the ammonia solution isremoved and evaporated to dryness under reduced pressure. The residue isdissolved in 1 ml water and filtered through a 0.45 μm syringe filter.The trityl protected hydrazide oligo is purified by reverse phase HPLCusing a Merck LiChrospher RP 18, 10 μM, column (analytical: 4×250 mm,flow=1.0 ml/min; preparative: 10×250, flow=3.0 mL/min) using 0.1 Mtriethylammonium acetate pH=7.0 (TEAA) as buffer A and 75% acetonitrilein buffer A as buffer B. A gradient of 0% B to 100% B in 100 min is usedfor analytical and preparative separations. The fractions containing thetrityl-on product were pooled and evaporated to dryness.

For the removal of the trityl protecting group the oligo is treated with80% acetic acid for 30 min at RT. The acid is removed in vacuo, and theresidue is dissolved in water then extracted twice with ethyl acetate.The aqueous layer is dried again and re-dissolved. Analytical HPLCusually shows a single product (is some cases as double peak) which canbe employed for further reactions without purification. AlternativelyHPLC purification can be performed using the solvent system describedabove.

Experiment 1.7 In Situ Generation of Hydrazide Functionality Synthesisof Oligonucleotides Using Phosphoramidites Containing Precursor Forms(e.g., Esters Such as Compound 1b FIG. 9B, Scheme 2: See Also FIG. 17B)

Oligonucleotides are synthesized using solid phase phosphoramiditechemistry on an automated oligonucleotide synthesizer. Thephosphoramidite with the precursor form of the hydrazide is applied as0.1 M solution in acetonitrile and coupled at the desired location inthe sequence using standard activating reagents and coupling times. Theuse of a phosphoramidite that contains a hydroxyl group labeled with anacid-labile protecting group as well as a hydrazide precursor allows theintroduction of the hydrazide at any position of the oligonucleotidebecause the precursor form of the hydrazide is stabile to the conditionsof the oligonucleotide synthesis while the reactive hydrazide is notformed until incubation with hydrazine.

The CPG bound oligo (1 mmol) is treated with a solution of 50 mgdiethylamine in 3.5 mL dichloromethane. After incubation overnight(light exclusion) the supernatant is removed and the support bound oligois washed several times with dichloromethane and dried in vacuo.

For the cleavage of the benzoyl and isobutyryl protecting groups theconversion of the ester at the 5′-end of the oligo to a hydrazide, andthe cleavage of the oligo from the support (FIG. 17B), the CPG with thebound oligo is treated with 1 ml 24% hydrazine hydrate. After 18 h underconstant agitation at 4° C. the reaction is complete. The isolation ofthe oligo from the hydrazine solution can be achieved by reversed phaseextraction (e.g. Sep-Pak or HPLC).

A C18 Sep-Pak cartridge (0.5 g Waters, No. 20515) is activated byrinsing with 10 mL acetonitrile and then 10 mL 0.1 M triethylammoniumbicarbonate buffer pH 7.0 (TEAB). The hydrazine solution is diluted withthe 5-fold volume of TEAB and applied to the cartridge. After binding ofthe oligo to the Sep-Pak column the residual hydrazine is washed awaywith 10 mL TEAB. The oligo is then eluted from the column withTEAB/acetonitrile (1:2). Oligo containing fractions are pooled andevaporated to dryness. For the RP-HPLC characterization and purificationof the product the same conditions as described in protocol 1 can beapplied.

Other examples are provided below wherein oligomers are processed tobecome linked to the multiple attachment moieties of the invention. Theoligos are numbered in sequence of their respective description in thisdisclosure.

Example 2 Experiment 2.1 Synthesis of Nonbranched Oligonucleotides 2.1.1Oligo 9: Hydrazide-15mer: (1-TTT TTT TTT TTT TTT-3′)

The synthesis and deprotection was performed as described with amiditecompound 1a. The trityl ON product elutes at 42.2 min under theconditions described. Oligo 9 elutes at 25.6 min (double peak). LRMS(ESI): M calc.: 4709.15, obs.: 4709.5.

2.1.2 Oligo 10: Hydrazide 19mer: (1-dGA TGA GCA GTT CTA CGT GG-3′)

The synthesis and deprotection was performed as described with amiditecompound 1a. The trityl ON product elutes at 41.5 min under theconditions described. Oligo 10 elutes at 25.1 min (single peak). HRMS(ESI): M calc.: 6092, obs.: 6092.

2.1.3 In Situ Generation of Hydrazides (Oligo 11: Hydrazide 19mer (8-dGATGA GCA GTT CTA CGT GG-Cy3)

The synthesis of the oligonucleotide was performed as describedpreviously. A CPG support loaded with Cy3 dye was used to label thefluorophor at the 3′ end of the oligo. The CPG bound oligo was treatedas outlined in Example 1 (E) above and the product was purified byRP-HPLC. The hydrazide oligo elutes at 31.8 min under the HPLCconditions described in Example 1 (D). LRMS (ESI): M calc.: 6599.7,obs.: 6598±2.

Experiment 2.2 Synthesis of Branching Oligonucleotides

For the introduction of multiple hydrazides into oligonucleotides,branching phosphoramidites, phosphoramidites having more than one estergroup which are converted into hydrazides, as well as a combination ofboth approaches were used. This flexible strategy allows the synthesisof oligonucleotides carrying defined numbers between one and up toseveral (˜40) hydrazides. The experiments herein are described usingp-RNA and are applicable to other oligonucleotides such as DNA.

Experiment 2.2.1 Synthesis of p-RNA Oligonucleotides

The synthesis of p-RNA oligonucleotides is performed as described in:Miculka, C.; Windhab, N.; Brandstetter, T. Burdinski, G; PCT patentapplication No. WO 99/15540 (1999) with the following exceptions andmodifications: Phosphoramidites of pentopyranosyl nucleosides are driedin vacuo over KOH and dissolved in dry acetonitrile to give a 0.1 Msolution. This solution is dried over freshly activated molecular sieve(3 Å) for 3 h and then applied for solid phase oligonucleotide synthesison a PE Biosystems Expedite 8905 DNA synthesizer. Other phosphoramiditesare dissolved at 0.1 M in dry acetonitrile and used without furthertreatment. For p-RNA oligonucleotides carrying a Cy3 dye at the 2′-end aCPG support custom loaded with monomethoxytrityl protected Cy3 (CAS:182873-80-9, AP-Biotech, Freiburg, Germany) a 0.1 M solution ofanhydrous pyridinium hydrochloride in dry acetonitrile is used asactivator. The detritylation time for pentopyranosyl nucleosides isincreased to 10 minutes and the coupling time is increased to 25minutes. All other reagents and solutions and procedures are accordingto the recommendation of the instrument manufacturer.

Experiment 2.2.2 Deprotection of p-RNA Oligonucleotides

For the cleavage of the β-cyanoethyl protecting groups theoligonucleotide is treated with a 1.5% (w/v) solution of diethylamine indichloromethane overnight at RT (light exclusion). The supernatant isremoved and the support bound oligonucleotide is washed several timeswith dichloromethane and dried in vacuo.

For the cleavage of the benzoyl and isobutyryl protecting groups, theconversion of the esters at the 5′-end of the oligo to hydrazides, andthe cleavage of the oligo from the support, the CPG with the bound oligois treated with 1 ml 24% hydrazine hydrate. After 18 h under constantagitation at 4° C. the reaction is complete. The isolation of the oligofrom the hydrazine solution can be achieved by reversed phase extraction(e.g. Sep-Pak or HPLC).

A C18 Sep-Pak cartridge (0.5 g Waters, No. 20515) is activated byrinsing with 10 mL acetonitrile and then 10 mL 0.1 M triethylammoniumbicarbonate buffer pH 7.0 (TEAB). The hydrazine solution is diluted withthe 5-fold volume of TEAB and applied to the cartridge. After binding ofthe oligo to the Sep-Pak column the residual hydrazine is washed awaywith 10 mL TEAB. The oligo is then eluted from the column withTEAB/acetonitrile (1:2). Oligo containing fractions are pooled andevaporated to dryness. The characterization and purification of theproducts is achieved by reverse phase HPLC using a Merck LiChrospher RP18, 10 μM, column (analytical: 4×250 mm, flow=1.0 ml/min; preparative:10×250, flow=3.0 mL/min) using 0.1 M triethylammonium acetate pH=7.0(TEAA) as buffer A and 75% acetonitrile in buffer A as buffer B. Agradient of 0% B to 100% B in 100 min (HPLC method A) or 30 min (HPLCmethod B) is used for analytical and preparative separations.

A. Oligo 12: Cy3 Labeled p-RNA Oligo with 1 Hydrazide: p-RNA Oligo4′-(Hyd₁) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1b.

B. Oligo 13: Cy3 Labeled p-RNA Oligo with 3 Hydrazides: p-RNA Oligo4′-(Hyd₃) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1d. The product elutes at 37.9 min (HPLC method A) under theconditions described. LRMS (ESI): M calc.: 3516.6, obs.: 3515.

C. Oligo 14: Cy3 Labeled p-RNA Oligo with 4 Hydrazides: p-RNA Oligo4′-(Hyd₂)₂ (SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1c and with symmetric branching phosphoramidite (SBA; Clontech,No. 5252-2). The product elutes at 37.3 min (HPLC method A) under theconditions described. LRMS (MALDI): M calc.: 3784.7, obs.: 3784

D. Oligo 15: Cy3 Labeled p-RNA Oligo with 8 Hydrazides: p-RNA Oligo4′-(Hyd₂)₄ (SBA)₂ (SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1c and with symmetric branching phosphoramidite (SBA; Clontech,No. 5252-2). The product elutes at 36.9 min (HPLC method A) under theconditions described. LRMS (MALDI): M calc.: 4661.1, obs.: 4464

E. Oligo 16: Cy3 Labeled p-RNA Oligo with Spacer and 8 Hydrazides: p-RNAOligo 4′-(Hyd)₄ (SBA)₂ (SBA) (S18) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1c and with symmetric branching phosphoramidite (SBA; Clontech,No. 5252-2) and Spacer 18 (S18, Glen research No. 10-1918-02). Theproduct elutes at 38.7 min (HPLC method A) under the conditionsdescribed.

F. Oligo 17: Cy3 Labeled p-RNA Oligo with 16 Hydrazides: p-RNA Oligo4′-(Hyd₂)₈ (SBA)₄ (SBA)₂ (SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described with amiditecompound 1c and with symmetric branching phosphoramidite (SBA; Clontech,No. 5252-2). The product elutes at 38.7 min (HPLC method A) under theconditions described.

G. Oligo 18: p-RNA Oligo with 4 Hydrazides (without Cy3 Dye): p-RNAOligo 4′-(Hyd₂)₂ (SBA) TAG GCA TT-2′

The synthesis and deprotection was performed as described with amiditecompound 1c. The product elutes at 12.75 min (HPLC method B) under theconditions described. LRMS (ESI): M calc.: 3275.1, obs.: 3275.4.

Experiment 2.3 General Procedure for the Conversion of HydrazideOligonucleotides into Boronate Oligonucleotides

50 nmol hydrazide oligonucleotide are dissolved in 200 μL 10 mM ammoniumacetate buffer pH 4.0 and 15 equivalents of 4-Formylphenlyboronic acid(Aldrich No. C43, 196-6; CAS: 87199-17-5) per hydrazide are added. Foran oligonucleotide containing 4 hydrazides for example 30 μL of a 0.1 Msolution of 4-Formylphenlyboronic acid in DMSO (3 μmol) are used. Themixture is incubated at RT for 1 h, 20 equivalents NaCNBH₃ per4-Formylphenlyboronic acid are added and incubation is continued for oneother hour at RT. For example for the oligonucleotide with 4 hydrazides150 μL (150 μmol) of a 1 M solution of NaCNBH₃ in 10 mM ammonium acetatebuffer pH 4.0 (6.3 mg dissolved in 1 mL) are necessary.

The removal of excess 4-Formylphenlyboronic acid and SodiumCyanoborohydride are removed by means of HPLC, gel filtration (PharmaciaPD 10 columns), or solid phase extraction (Merck LiChrolute columns).For boronate modified oligonucleotides it is crucial to use an endcappedHPLC column. Typical conditions are 5 μm Phenomenex Luna Phenyl Hexylcolumns (analytical: 4.6×250 mm, flow=1.0 ml/min; preparative: 10×250,flow=3.0 mL/min) using 0.1 M triethylammonium acetate pH=7.0 (TEAA) asbuffer A and 75% acetonitrile in buffer A as buffer B. A gradient of 0%B to 100% B in 100 min (HPLC method A) or 30 min (HPLC method B) is usedfor analytical and preparative separations. Product containing fractionsare pooled and evaporated to dryness.

A. Oligo 19: p-RNA Oligo with 1 Boronate: p-RNA Oligo 4′-(PBA) TAG GCATT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 12 as starting material.

B. Oligo 20: p-RNA Oligo with 3 Boronates: p-RNA Oligo 4′-(PBA)₃ TAG GCATT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 13 as starting material.

C. Oligo 21: p-RNA Oligo with 4 Boronates: p-RNA Oligo 4′-(PBA)₄ (SBA)TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 14 as starting material.

D. Oligo 22: p-RNA Oligo with 8 Boronates: p-RNA Oligo 4′-(PBA)₈ (SBA)₂(SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 15 as starting material. The productelutes at 46.3 min (HPLC method A) under the conditions described.

E. Oligo 23: p-RNA Oligo with Spacer18 and 8 Boronates: p-RNA Oligo4′-(PBA)₈ (SBA)₂ (SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 16 as starting material.

F. Oligo 24: p-RNA Oligo with 16 Boronates: p-RNA Oligo (PBA)₁₆ (SBA)₄(SBA)₂ (SBA) TAG GCA TT (Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 17 as starting material. The productelutes at 49.0 min (HPLC method A) under the conditions described.

G. Oligo 25: p-RNA Oligo with 1 Boronate: p-RNA Oligo 4′(PBA)-TAG GCA TT(Cy3)-2′

The synthesis and deprotection was performed as described in the generalprotocol using oligonucleotide 18 as starting material.

Example 3 HPLC Analysis

Upon completion of the synthesis of hydrazide oligos, the first set ofexperiments examined the solution reaction kinetics of a hydrazidelabeled oligo with an NHS or Sulfo-NHS ester. To a solution of 5 uL of132 uM hydrazide ATA5 in 30 uL of 50 mM histidine was added 5 uL of 10mM NHS acrylate. The solution was stirred at RT for a short period oftime then injected into an HPLC system. The HPLC trace of the compoundsin the solution indicated the quantities of hydrazide ATA5 andN′acrylo-ATA5 dihydrazide present in the reaction mixture for a givenreaction time. The retention times of the starting ATA5 hydrazide andthe modified ATA5 hydrazide were distinct and separable.

FIGS. 10A-C show three separate traces of the reaction mixture. Thefirst trace (A) was obtained from an unmodified ATA5 hydrazide (A), andthe third trace (C) represents a completely modified ATA5 hydrazide (B)after a reaction time of 5 minutes with NHS acrylate. The middle trace(B) represents an incomplete modification captured 1 minute into thereaction. Given the approximate consumption of ATA5 hydrazide, apseudo-first order reaction rate of 1200 M⁻¹s⁻¹ is determined.

Comparison of this rate to other attachment systems utilized is shown inFIG. 11. The reaction rate for an NHS ester with a hydrazide in anaqueous environment represents an exceptionally efficient reaction.Furthermore, the pH of the reaction was altered to determine the pHdependence of the hydrazide modification. Experiments were carried outwith a buffering system of 50 mM histidine adjusted with HCl to pH=6,5.5, 5.0, 4.5, and 4. The transformation continued down to pH=4.5.However, at pH=4, the hydrazide oligo was unaffected, constituting notransformation and therefore a pH lower limit of approximately 4.5.

Example 4 Chip Preparation

Microarray containing chips are plasma cleaned 5 minutes under Argon.The 25 site 1 cm by 1 cm chips are then silanized using vapor phasedeposition. To the center of the microarray is added 0.10 uL of a 20%(by mass) solution of 9:1 (molar ratio) acrylamide/bisacrylamide in 1:1DMSO/H₂O with 0.3% Daracur 4265 as a UV initiator. The chip is placedinto a microreaction molding system to which the microarray site ispressed to a UV window containing a square 4 uM cavity, 3.mm on a side.The solution is irradiated for 20 sec with UV light, removed from themolding system, rinsed with water and air dried. The well forms a squarehydrogel layer over the microarray. Excess polymerization, beyond theparameters of the mold, is removed.

To the existing permeation layer is added 0.80 uL of a solutioncontaining 20% (by mass) monomer concentration of NHS orSulfo-NHS/Am/Bis 10/83/7 (molar ratio) and allowed to saturate theexisting polymer for 1 minute. The chip is loaded onto the microreactionmolding system and polymerized as above with a circular mold with adiameter of 4.6 mm and a well depth of 5 uM. This second mold completelyencompasses and extends beyond the existing square layer. Attachment ofthe second layer is accomplished through intercalation of polymer chainsand bind silane. The chips are washed with water and dried withcompressed air and subsequently tested in the following experiments.

Experiment 4.1 Activated Ester Concentration; Labeled Capture Address

To chips modified with the two fold permeation layer as described abovecontaining 0, 1, 2 and 4% Sulfo-NHS was electronically loaded 500hydrazide-T12-BTR as a specific labeled capture while 50 mM nMbiotin-T12-BTR was used as a nonspecific labeled capture. All solutionswere buffered in 50 mM histidine. Captures were addressed at a currentof 500 nA/pad for 120 seconds, 4 pads at a time. Each chip was washedwith 1% SDS, 0.2×STE and soaked in 1% SDS for 20 minutes. The chips wereimaged for 1 second and the average MFI values were recorded.

As can be seen by in FIG. 12, the covalent attachment of the labeledhydrazide oligo is dependent on the amount of activated ester in thepermeation layer and increases as the concentration increases. Thenonspecific attachment of a biotin labeled oligo is also quite low,averaging 40 MFI/s for the experiment.

Experiment 4.2 Electronic Conditions

To chips modified with the two fold permeation layer as described abovecontaining 10% NHS or Sulfo-NHS was electronically loaded 500 and 5 nMhydrazide-T12-BTR as a specific labeled capture. 500 mM nMbiotin-T12-BTR was used as a nonspecific labeled capture. All solutionswere buffered in 50 mM histidine. Captures were addressed at currents of400, 500, 600, 700 and 800 nA/pad for 120 seconds, 3 pads at a time.Nonspecific captures were loaded at 800 nA/pad. Each chip was washedwith 1% SDS, 0.2×STE and soaked in 1% SDS for 20 minutes. The chips wereimaged for 1 second and the average MFI values were recorded.

As can be seen by in FIG. 13, attachment of a specific capture to theNHS modified permeation layer dramatically increases at 600 nA, whileSulfo-NHS modified hydrogels required a slightly higher current formaximum attachment (FIG. 14).

Experiment 4.3 Effect of Multiple Binding

To chips modified with the two fold permeation layer as described abovecontaining 10% NHS were loaded Cy3 labeled ATA5 oligos containing 1, 2,4, or 8 hydrazide moieties. The four oligomers were electronicallyaddressed at 500 nM with a current of either 700 or 800 nA/pad for 120s, buffered in 50 mM histidine. Upon completion, the chips were washedand the binding levels were measured.

The recorded MFI/s values are displayed in FIG. 15. A comparison thenumber of hydrazide moieties available for attachment per oligomer givenequal currents indicates an increased binding level with the increase inhydrazides to the oligomer.

Experiment 4.4 Reverse Dot-Blot Electronic Hybridization

To chips modified with the two fold permeation layer as described abovecontaining 15% NHS was loaded an octa-hydrazide ATA5 oligomer with a Cy3label as a specific capture. The specific capture was loaded at 500 nMwith a current of either 600 or 700 nA/pad for 120 s, buffered in 50 mMhistidine. Electronic Hybridization was carried out with 5 nMRCA5-T12-Cy5 as a specific target while a solution of 5 nM RCA4-Cy5 wasused as a nonspecific target. The targets were loaded at 400 nA/pad for60 seconds, the chips were washed according to the standard protocol andimaged.

The data presented in FIG. 16 clearly indicates the hybridization of thespecific target preferentially to the nonspecific target. It should alsobe noted that in agreement with data reported above, the increase incurrent, from 600 to 700 nA for the electronic loading of the captureresults in an increase in the hybridization.

Example 5 Synthesis of Biomolecules Having Noncovalent Binding Moieties

FIGS. 4 and 5A and B depict the syntheses of oligonucleotides containingmultiple binding moieties. In FIG. 4, oligo synthesis is depictedwherein there is added a single branched phosphoramidite containing twoPBAs. FIGS. 5 A and B show two branches with four PBAs, and threebranches with eight PBAs, respectively. Syntheses as depicted werecarried out on an ABI394 DNA Synthesizer. The stepwise coupling yield ofbranched phosphoramidite was similar to the regular nucleotidephosphoramidites, about 96-98%. The PBA phosphoramidite was applied atthe last step. The cleaving of oligonucleotides from the solid supportand the removal of protecting groups were the same as the handling ofregular oligonucleotides as is well known to those of skill in the art.

The PBA-containing branched oligonucleotides were purified and analyzedby HPLC. The HPLC of PBA-containing oligonucleotide showed a broaderpeak than that of a regular oligonucleotide.

Experiment 5.1 Electronic Loading of Biomolecules Via Non-CovalentBinding Moieties

20 nM non-branched and branched PBA-containing ATA5 capture probes wereloaded on hydrogel substrates electronically. The capture probes wereloaded in 50 mM histidine, 10 pads at a time for 120 seconds. 20 nMRCA5-BTR was loaded passively for 5 minutes. The substrates were washedand imaged. Analysis showed that both branched and unbranched captureprobes were immobilized to the permeation layer, as desired.

Experiment 5.2 Stability of Electronically Loaded Biomolecules ViaNon-Covalent Binding Moieties

Oligos 20, 21 and 22 (p-RNAs containing 3, 4, and 8 PBA binding sites)were electronically addressed to SHA modified hydrogel chips. Uponcompletion, initial images were recorded after a standard washingprocedure previously described. The chip arrays were then subject toregular irrigation with repeated rinsing with 10 uL of 50 mM histidine.Images were recorded after 5 washings. The results shown in FIG. 21contain 2 features. Primarily the recorded signals for the higher orderdendrimers which have a higher number of attachment sites per oligo isdistinctly higher. Also, the signal is quite stable over a period of 25wash cycles illustrating the improved stability of the use ofdendrimeric attachment systems. Oligo 22 has lost approximately 14% ofits initial signal while oligos 20 and 21 have decreased 25 and 35%respectively.

Example 6 Covalent Attachment Via Multiple Hydrazone Formation

Previously, oligos modified with a single amine or hydrazide have beenelectronically loaded onto aldehyde modified hydrogels. The interactionof an aldehyde with an amine or hydrazide results in the formation of animine (carbon with a double bond to nitrogen) or a hydrazonerespectively. These moieties are reversible under aqueous conditions andrequire further reduction with NaBH₃CN to form a stable irreversiblecovalent attachment. Indeed, electronic concentration of an oligomercontaining a single hydrazide resulted in attachment of the oligomer tothe surface via hydrazone formation. Elimination of the reduction stepresulted in a readily hydrolyzed and unstable linkage in which the boundoligo readily diffused away. The use of dendrimeric hydrazides providesa means of covalent attachment through a somewhat unstable linkage whichdoes not require further reduction; provided there are a significantnumber of hydrazones formed per oligo. The reversible hydrazoneformation can occur with some linkage sites while others remain intact(FIG. 22). The hydrazide is incapable of diffusion, and trapped withinan aldehyde rich environment, can readily reform. This equilibrium takesadvantage of the increased number of attachment sites per oligo and,provided all linkages do not hydrolyze at once, is contemplated toprovide a stable attachment system. Aldehyde rich permeation layers canbe prepared directly, as in glyoxyl agarose, or can be obtained from anacetal modified permeation layer. In the latter, the acetal moiety isreadily hydrolyzed in the presence of acid to afford an aldehyde. Theacetal serves as a protecting group, preserving the aldehydefunctionality until activation is desired. Hydrolysis can be completedwith exposure to an acidic solution for 1 hr or subjected to a mildelectronic current buffered in a dilute salt solution. The latter methodprovides site specific hydrolysis by taking advantage of the acidgenerated at the cathode.

Experiment 6.1 Dendrimeric Hydrazide Oligomers Attached to GlyoxylAgarose

Standard 25 site chips were spin coated with glyoxyl agarose (FMC,Princeton, N.J.)). 500 nM Hydrazide Cy3 labeled oligos containing 1, 2,4, and 8 hydrazides were electronically loaded at 500 nA/pad for 2minutes each, buffered in 50 mM histidine. The chips were washedaccording to established procedure and imaged. The recorded MFI/s valuesare displayed in FIG. 23. The oligos with one or two hydrazides werequite unstable and as expected afforded little or no detectablefluorescence beyond the background noise. The oligos with a highernumber of hydrazides are capable of forming a stable covalentattachment.

Experiment 6.2 Dendrimeric Hydrazide Oligomers Attached to AcetalModified Hydrogels: Deprotection and Covalent Attachment

Standard 25 array site microchips were modified with a single layerhydrogel composed of acrylamide, bisacrylamide and vinyl acetal in a15:2:3 ratio. Selected sites were predisposed to a current of 300 nA/padfor 2 minutes in a 50 mM NaCl solution to hydrolyze the acetalfunctionality exposing the aldehydes. Dendrimeric hydrazide oligomerscontaining 8 hydrazides per oligo were electronically loaded at 500nA/pad for 2 minutes buffered in 50 mM histidine to pads which had beenactivated and to those that had not. A nonspecific oligo was alsoelectronically loaded onto both acetal and aldehyde modified sites.After a standard wash cycle, the chips were imaged. The recorded MFI/sdata is displayed in FIG. 24.

As can be seen in FIG. 24, pads which had been electronically activated,then loaded electronically with a dendrimeric labeled oligomer exhibitthe highest fluorescence signal. Interestingly, those pads which werenot pre-addressed, remaining as acetals also indicate some attachment ofhydrazide modified oligomers. Presumably, the electronic current appliedto concentrate the oligomer generated enough acid to surpass thebuffering capacity of histidine locally and was therefore able tohydrolyze a significant quantity of acetal moieties.

Example 7 Coupling of Hydrazide Oligonucleotides to Molecules Other thanSubstrate Surfaces Experiment 7.1 Reaction of Hydrazide-15mer 9 withBenzyloxy Acetaldehyde; FIG. 19

10 μmol Hydrazide Oligonucleotide 9 are dissolved in 60 μL 10 mMammonium acetate buffer (pH 4.0). 1 drop benzyloxyacetaldehyde (CAS:6065-87-3; C9H10O2 [150.1760] Aldrich No. 38, 218-3) is added and themixture is allowed to stand at RT for 1 h. The solvent and excess ofaldehyde is removed in vacuo and the product is analyzed by HPLC(Column: Merck LiChrospher RP 18, 10 μM, 4×250 mm; Buffer A=0.1 Mtriethylammonium acetate pH=7.0, Buffer B=75% acetonitrile in buffer A;Flow=1.0 mL/min; Gradient: 0% B to 100% B in 100 min). The retentiontime of the product is 30.7 min, oligo 9 elutes at 25.5 min.

Experiment 7.2 Conjugation Reaction of Oligo 10 with a Peptide, FIG. 20

4.4 nmol Oligo 10 are dissolved in 60 μL 10 mM ammonium acetate buffer(pH 4.0). 44 nmol (10 eq.) antipain hydrochloride (CAS: 37682-72-7;C27H44N10O6.2 HCl; [677.6304]; Calbio No. F 178220) in 15 μL buffer areadded and agitated 3 h at RT. The intermediate product is reduced withNaBH₃CN (100 eq.) for 1 h at RT. The product is isolated by HPLC(Column: Merck LiChrospher RP 18, 10 μM, 4×250 mm; Buffer A=0.1 Mtriethylammonium acetate pH=7.0, Buffer B=75% acetonitrile in buffer A;Flow=1.0 mL/min; Gradient: 10% B to 85% B in 60 min). The retention timeof the product (oligonucleotide peptide conjugate) is 16.5 min, oligo 10elutes at 13.9 min. MS (ESI): calc: 6680.6; obs.: 6679.6)

Example 8 Passive Application of Hydrazide Modified Biomolecules onSlide Surfaces

For the binding of hydrazide modified oligonucleotides to commerciallyavailable slides a series of p-RNA oligonucleotides containing 1 to 16hydrazides were used. Along with oligonucleotides 12, 13, and 14,oligomers with 3 and 6 hydrazides, prepared from 1d, were used.Additionally, an amine terminated oligomer (prepared with 5′ AminoModifier C6; Glenn Research) and an oligonucleotide without modificationare used as nonspecific controls. All oligomers are labeled with Cy3 atthe 2′ end and retain the same nucleotide sequence.

Experiment 8.1 Attachment to Surmodics 3D Link™ Amine Binding Slides

Oligonucleotides are dissolved in 3D Link™ print buffer (Surmodics, Inc,Eden Prairie, Minn.) at pH=8.5 with concentrations ranging between 10 μMand 100 nM. From each solution, 0.5 μL was applied directly to the slidesurface and incubated at room temp. in a sealed chamber above asaturated NaCl solution overnight in the dark. The slides are thentreated for 15 min at 50° C. with 3D Link™ blocking buffer to blockunreacted surface sites. The slides were washed twice with waterfollowed by a 30 min wash with 0.2% SDS at 50° C. and finally two waterwashings, then allowed to air dry. The fluorescence detection waspreformed on a Pharmacis scanner with 20 second integration times.Images as well as intensity profiles are displayed in FIG. 25.

The nonspecific oligonucleotide afforded a signal between 10×10³ and25×10³ relative units at 10 μM. The signal compares in intensity withthat observed for an oligonucleotide containing a single amino group. Incontrast, the hydrazide modified oligonucleotide affords a much higherloading of 35-40×10³ fluorescence units. Further, the hydrazide modifiedoligonucleotide has a higher fluorescence signal at lowerconcentrations, with a lower limit of detection of 1.25 μM, as comparedto the amine modified oligomer which has a lower detection limit of 5μM.

Experiment 8.2 Attachment to SuperAldehyde Slides

Oligonucleotides are dissolved in either Surmodics 3D Link™ print bufferat pH=8.5 with concentrations ranging from 10 μM to 100 nM or in 10 mMammonium acetate buffer at pH=4.0. From each solution, 0.5 μM areapplied to the surface of SuperAldehyde slides (Telechem International,Inc Sunnyvale, Calif.) and allowed to incubate overnight at rt. Theslides are then treated twice with 0.2% SDS and washed 4 times withwater (2 min each). The surface was then treated with a solution of 0.3%NaBH₃CN in PBS buffer, pH=7, with 133 mL ethanol to eliminate bubbling.This was followed by three 1 min washings with 0.2% SDS and water.Fluorescence detection was preformed on a Pharmacis scanner with 20 sintegration times. Images as well as intensity profiles are displayed inFIG. 26.

As can be seen if FIG. 26, at both pH=8.5 and 4.0 the hydrazideoligonucleotide affords a much higher signal intensity as compared tothe amine terminated oligomer and is unaffected by the change in pH.Furthermore, given the same concentrations, the hydrazide modifiedoligomer affords much higher signal intensity than the amine modifiedoligomers. The amine oligonucleotides are no longer detectable below 2.5μM while the hydrazide oligomers are detected as low as 1.25 μM.

The foregoing is intended to be illustrative of the embodiments of thepresent invention, and are not intended to limit the invention in anyway. Although the invention has been described with respect to specificmodifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare to be included herein. All publications and patent applications areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method of binding a biomolecule to a substrate comprising the stepsof: providing a biomolecule; contacting the biomolecule with a branchedlinking moiety to form a branched linking structure that is adapted tocouple with the substrate, wherein the branched linking structure hasmultiple reactive groups; and contacting the branched linking structurewith the substrate to form a coupled substrate binding structure, wherethe biomolecule is bound to the substrate through multiple linkages. 2.The method of claim 1, further comprising the step of activating thebranched linking structure before contacting the branched linkingstructure with the binding moiety.
 3. The method of claim 1, wherein thesubstrate is a polymer.
 4. The method of claim 1, wherein the substrateis an activated polymer.
 5. The method of claim 1, wherein the substrateis in contact with an electronically addressable microchip.
 6. Themethod of claim 1, wherein the substrate is an activated glass slide. 7.The method of claim 1, wherein the substrate is a glass slide.
 8. Themethod of claim 1, wherein the biomolecule comprises a Lewis base or anucleophile.
 9. The method of claim 1, wherein the biomolecule comprisesa Lewis acid or an electrophile.
 10. The method of claim 8, wherein theLewis base or the nucleophile is selected from the group consisting ofalcohol, amine, hydrazine, hydrazide, salicylic hydroxamic acid, andsulfhydryl.
 11. The method of claim 9, wherein the Lewis acid or theelectrophile is selected from the group consisting of an epoxide,aziridine, vinyl, aldehyde, ketone, acetal, disulfide, carboxylic acid,amide, bromo or iodoacetamide, N-hydroxysuccinimidyl ester,sulfo-N-hydroxysuccinimidyl ester, azlactone, isocyanate,thioisocyanate, phenyl boronic acid, and carbonate.
 12. The method ofclaim 1, wherein the branched linking moiety comprises aphosphoramidite.
 13. The method of claim 12, wherein the phosphoramiditeis selected from the group consisting of Diethyl3-[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]glutarate; Diethyl5-{[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]methyl}isophthalate;Dimethyl3,3′-(2-{[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]methyl}-2-{[2-(methoxycarbonyl)ethoxy]methyl}propane-1,3-diylbisoxy)dipropionate;Ethyl 6-[(2-cyanoethoxy)(diisopropylamino)phosphanyloxy]hexanoate; and6-[(2-Cyanoethoxy)(diisopropylamino)phosphanyloxy)-N′-tritylhexanohydrazide.14. The method of claim 1, wherein the multiple linkages are covalentbonds.
 15. The method of claim 1, wherein the multiple linkages arenon-covalent bonds.